Despite recent advances in vascular biology, the complex process of vasculogenesis is still poorly understood. It has become increasingly apparent that the process of vasculogenesis observed during ontogeny may be recapitulated in the context of tissue injury and certain disease states during adulthood. For example, pluripotent progenitor cells have the ability to differentiate into vascular smooth muscle cells during the pathogenesis of vascular disease. However, the regulatory pathways that govern vascular smooth muscle cell (VSMC) lineage commitment and differentiation during development and vascular diseases remain to be further defined. Further insight is needed to identify and characterize the transcriptional pathways and target genes involved in VSMC differentiation to provide improved treatment modalities to patients with vascular disease. Based on previous evidence that the Iroquois homeobox (Irx) proteins are critical factors in a number of developmental processes, we have begun to test the hypothesis that Irx transcription factors are downstream mediators of the effect of Notch on VSMC differentiation in various cell- based model systems which include the human embryonic stem cell line hESBGN-02 (NIH Code: BG02). Indeed, our preliminary data indicates that Irx3 is expressed in VSMCs and is modulated in a temporal manner during VSMC differentiation from multipotent progenitor cells in vitro. Moreover,, the inhibition of Notch signaling up-regulates Irx3 expression in association with VSMC differentiation in the P19 model system. The overall goal of this proposed project is utilize well,established in vitro models that simulate the process of vasculogenesis to define the role of Irx3 during the process of VSMC differentiation The central hypothesis of this proposal is that the Iroquois Homeobox transcription factor Irx3 is essential in early vascular smooth muscle cell differentiation. Moreover, that Irx3 directly modulates the expression- of essential target genes which promote early VSMC lineage commitment. Thus, the specific aims are to: Aim 1: Identify and characterize the temporal relationship of Irx3 during the process of VSMC differentiation in vitro and in vivo. Aim 2: Define the essential role of Irx3 as downstream effectors of the Notch signaling pathway during early VSMC lineage commitment. Aim 3: Determine the Irx3 responsive target genes associated with early VSMC lineage commitment. (End of Abstract)